Selective estrogen receptor-beta ligands

ABSTRACT

The invention relates to novel compounds having the general formula: (I) and arc useful as selective ER-β ligands in the treatment or prophylaxis of Alzheimer&#39;s disease, anxiety disorders, depressive disorders, osteoporosis cardiovascular disease, rheumatoid arthritis or prostate cancer.

TECHNICAL FIELD

The present invention is directed to a series of ligands, and moreparticularly to estrogen receptor-β ligands which have betterselectivity than estrogen for the estrogen receptor-β over the estrogenreceptor-α, as well as to methods for their production and use in thetreatment of diseases related to the estrogen receptor-β, specifically,Alzheimer's disease, anxiety disorders, depressive disorders (includingpost-partum and post-menopausal depression), osteoporosis,cardiovascular disease, rheumatoid arthritis, or prostate cancer.

BACKGROUND

Estrogen-replacement therapy (“ERT”) reduces the incidence ofAlzheimer's disease and improves cognitive function in Alzheimer'sdisease patients (Nikolov et al. Drugs of Today, 34(11), 927-933(1998)). ERT also exhibits beneficial effects in osteoporosis andcardiovascular disease, and may have anxiolytic and anti-depressanttherapeutic properties. However, ERT shows detrimental uterine andbreast side effects that limit its use.

The beneficial effects of ERT in post-menopausal human women is echoedby beneficial effects of estrogen in models relevant to cognitivefunction, anxiety, depression, bone loss, and cardiovascular damage inovariectomized rats. Estrogen also produces uterine and breasthypertrophy in animal models reminiscent of its mitogenic effects onthese tissues in humans.

The beneficial effects of ERT in post-menopausal human women is echoedby beneficial effects of estrogen in models relevant to cognitivefunction, anxiety, depression, bone loss, and cardiovascular damage inovariectomized rats. Specifically, experimental studies havedemonstrated that estrogen effects the central nervous system (“CNS”) byincreasing cholinergic function, increasing neurotrophin/neurotrophinreceptor expression, altering amyloid precursor protein processing,providing neuroprotection against a variety of insults, and increasingglutamatergic synaptic transmission, among other effects. The overallCNS profile of estrogen effects in pre-clinical studies is consistentwith its clinical utility in improving cognitive function and delayingAlzheimer's disease progression. Estrogen also produces mitogeniceffects in uterine and breast tissue indicative of its detrimental sideeffects on these tissues in humans.

The estrogen receptor (“ER”) in humans, rats, and mice exists as twosubtypes, ER-α and ER-β, which share about a 50% identity in theligand-binding domain (Kuiper et al. Endocrinology 139(10) 4252-4263(1998)). The difference in the identity of the subtypes accounts for thefact that some small compounds have been shown to bind preferentially toone subtype over the other (Kuiper et al.).

In rats, ER-β is strongly expressed in brain, bone and vascularepithelium, but weakly expressed in uterus and breast, relative to ER-α.Furthermore, ER-α knockout (ERKO-α) mice are sterile and exhibit littleor no evidence of hormone responsiveness of reproductive tissues. Incontrast, ER-β knockout (ERKO-β) mice are fertile, and exhibit normaldevelopment and function of breast and uterine tissue. Theseobservations suggest that selectively targeting ER-β over ER-α couldconfer beneficial effects in several important human diseases, such asAlzheimer's disease, anxiety disorders, depressive disorders,osteoporosis, and cardiovascular disease without the liability ofreproductive system side effects. Selective effects on ER-β-expressingtissues (CNS, bone, etc.) over uterus and breast could be achieved byagents that selectively interact with ER-β over ER-α.

It is a purpose of this invention to identify ER-β-selective ligandsthat are useful in treating diseases in which ERT has therapeuticbenefits.

It is another purpose of this invention to identify ER-β-selectiveligands that mimic the beneficial effects of ERT on brain, bone andcardiovascular function.

It is another purpose of this invention to identify ER-β-selectiveligands that increase cognitive function and delay Alzheimer's diseaseprogression.

SUMMARY OF THE INVENTION

This present invention is directed to compounds having the genericstructure:

These compounds are ERG-β-selective ligands, which mimic ERT, but lackundesirable side effects of ERT and are useful in the treatment orprophylaxis of Alzheimer's disease, anxiety disorders, depressivedisorders, osteoporosis, cardiovascular disease, rheumatoid arthritis orprostate cancer.

These compounds particularly satisfy the formula:(K _(iαA) /K _(iβA))/(K _(iαE) /K _(iβE))>1,preferably:(K _(iαA) /K _(iβA))/(K _(iαE) /K _(iβE))>30,more preferably:(K _(iαA) /K _(iβA))/(K _(iαE) /K _(iβE))>100,wherein K_(iαA) is the K_(i) value for the ligand in ER-α; K_(iβA) isthe Ki value for the ligand in ER-β; K_(iαE) is the K_(i) value forestrogen in ER-α; and K_(iβE) is the K_(i) value for estrogen in ER-β.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the instant invention are ER-β-selective ligands of thestructure:

wherein:

-   -   R¹ is H, C₁₋₈alkyl, phenyl, or a 5- or 6-membered ring        heterocycle containing 1, 2 or 3 heteroatoms each independently        selected from O, N and S and additionally having 0 or 1 oxo        groups and 0 or 1 fused benzo rings, wherein the C₁₋₈alkyl,        phenyl or heterocycle is substituted by 0, 1, 2 or 3        substituents selected from —R^(a), —OR^(a), —SR^(a),        —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),        —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a),        —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano, nitro        and C₁₋₃haloalkyl;    -   R² is C₁₋₈alkyl, phenyl, —C(═O)phenyl, benzyl or a 5- or        6-membered ring heterocycle containing 1, 2 or 3 heteroatoms        each independently selected from O, N and S and additionally        having 0 or 1 oxo groups and 0 or 1 fused benzo rings, wherein        the C₁₋₈alkyl, phenyl, —C(═O)phenyl, benzyl or heterocycle is        substituted by 1, 2 or 3 substituents selected from —OR^(a),        —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),        —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),        —NR^(a)S(═O)₂R^(a); —C(═C)R^(a), —S(═O)R^(a), —S(═O)₂R^(a),        halogen, cyano, nitro and C₁₋₃haloalkyl; and wherein the phenyl,        —C(═O)phenyl, benzyl or heterocycle is additionally substituted        by 0, 1 or 2 substituents selected from C₁₋₆alkyl, phenyl or        benzyl;    -   R³ is hydrogen, C₁₋₆alkyl, —(CH₂)_(m)phenyl or        —(CH₂)_(m)heterocycle, wherein the heterocycle is a 5- or        6-membered ring heterocycle containing 1, 2 or 3 heteroatoms        each independently selected from O, N and S and additionally        having 0 or 1 oxo groups and 0 or 1 fused benzo rings;    -   R⁴ is H, halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, —(CH₂)_(m)phenyl or        —(CH₂)_(m)heterocycle, wherein the heterocycle is a 5- or        6-membered ring heterocycle containing 1, 2 or 3 heteroatoms        each independently selected from O, N and S and additionally        having 0 or 1 oxo groups and 0 or 1 fused benzo rings;    -   R⁵ is —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),        —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a),        —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a),        —S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl; or R⁵ is        C₁₋₃alkyl containing 1 or 2 substituents selected from —OR^(a),        —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),        —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),        —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a),        halogen, cyano and nitro;    -   R⁶ is —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),        —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a),        —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a),        —S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl;    -   R⁷ is —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),        —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a),        —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a),        —S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl;    -   R⁸ is —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),        —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a),        —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a),        —S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl; or R⁸ is        C₁₋₃alkyl containing 1 or 2 substituents selected from —OR^(a),        —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),        —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),        —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a),        halogen, cyano and nitro;    -   R⁹ is H, C₁₋₅alkyl or C₁₋₃haloalkyl;    -   R^(a) is H, C₁₋₆alkyl, phenyl or benzyl;    -   m is 0, 1, 2or 3; and    -   n is 0 or 1.

In one embodiment of the above compounds, R¹ is H, phenyl or a 5- or6-membered ring heterocycle containing 1, 2 or 3 heteroatoms eachindependently selected from O, N and S and additionally having 0 or 1oxo groups and 0 or 1 fused benzo rings, wherein the phenyl orheterocycle is substituted by 0, 1, 2 or 3 substituents selected from—R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —OC(═O)R^(a), —NR^(a)C(═O)R^(a),—NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —S(═O)R^(a), —S(═O);R^(a),halogen, cyano, nitro and C₁₋₃alkyl substituted with 1-7 halogen atoms.

In another embodiment of the above compounds, R² is phenyl,—C(═O)phenyl, benzyl or a 5- or 6-membered ring heterocycle containing1, 2 or 3 heteroatoms each independently selected from O, N and S andadditionally having 0 or 1 oxo groups and 0 or 1 fused benzo rings,wherein the C₁₋₈alkyl, phenyl, benzyl or heterocycle is substituted by1, 2 or 3 substituents selected from —OR^(a), —SR^(a), —NR^(a)R^(a),—OC(═O)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl; andwherein the phenyl, —C(═O)phenyl, benzyl or heterocycle is additionallysubstituted by 0, 1 or 2 substituents selected from C₁₋₆alkyl, phenyl orbenzyl.

In another embodiment of the above compounds, R³ is C₁₋₆alkyl,—(CH₂)_(m)phenyl or —(CH₂)_(m)heterocycle, wherein the heterocycle is a5- or 6-membered ring heterocycle containing 1, 2 or 3 heteroatoms eachindependently selected from O, N and S and additionally having 0 or 1oxo groups and 0 or 1 fused benzo rings.

In another embodiment of the above compounds, R⁴ is halogen, C₁₋₆alkyl,C₁₋₆haloalkyl, —(CH₂)_(m)phenyl or —(CH₂)_(m)heterocycle, wherein theheterocycle is a 5- or 6-membered ring heterocycle containing 1, 2 or 3heteroatoms each independently selected from O, N and S and additionallyhaving 0 or 1 oxo groups and 0 or 1 fused benzo rings.

In another embodiment of the above compounds, R⁵ is C₁₋₆alkyl, —OR^(a),—SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl; orR⁵ is C₁₋₃alkyl containing 1 or 2 substituents selected from —OR^(a),—SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano and nitro.

In another embodiment of the above compounds, R⁶ is —R^(a), —SR^(a),—NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl.

In another embodiment of the above compounds, R⁷ is —OR^(a), —SR^(a),—NR^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a),—NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a),—S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl.

In another embodiment of the above compounds, R⁸ is C₁₋₆alkyl, —OR^(a),—SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl; orR⁸ is C₁₋₃alkyl containing 1 or 2 substituents selected from —OR^(a),—SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R⁸, halogen, cyano and nitro.

In another embodiment of the above compounds, R⁶ is OH.

In another embodiment of the above compounds, R³ is H, R⁴ is H and R⁶ isOH.

Particularly useful compounds have any of the above embodiments and alsosatisfy the equation:(K _(iαA) /K _(iβA))/(K _(iαE) /K _(iβE))>100, wherein

-   -   K_(iαA) is the K_(i) value for the agonist in ER-α;    -   K_(iβA) is the K_(i) value for the agonist in ER-β;    -   K_(iαE) is the K_(i) value for estrogen in ER-α; and    -   K_(iβE) is the K_(i) value for estrogen in ER-β.

Another aspect of the invention is the use of any of the above compoundembodiments for the manufacture of a medicament for the treatment orprophylaxis of Alzheimer's disease, anxiety disorders, depressivedisorders, osteoporosis, cardiovascular disease, rheumatoid arthritis orprostate cancer.

Another aspect of the invention is the use of any of the above compoundembodiments in the treatment or prophylaxis of Alzheimer's disease,anxiety disorders, depressive disorders (including postpartum andpost-menopausal depression), osteoporosis, cardiovascular disease,rheumatoid arthritis or prostate cancer,

A pharmaceutical composition comprising:

Another aspect of the invention involves a pharmaceutical compositioncomprising a therapeutically-effective amount of a compound according toany of the above embodiments and a pharmaceutically-acceptable diluentor carrier.

C_(Y-Z)alkyl, unless otherwise specified, means an alkyl chaincontaining a minimum Y total carbon atoms and a maximum Z total carbonatoms. These alkyl chains may be branched or unbranched, cyclic, acyclicor a combination of cyclic and acyclic. For example, the followingsubstituents would be included in the general description “C₄₋₇alkyl”:

The term “oxo” means a double bonded oxygen (═O).

The compounds of the invention may contain heterocyclic substituentsthat are 5- or 6-membered ring heterocycles containing 1, 2 or 3heteroatoms each independently selected from O, N and S and additionallyhaving 0 or 1 oxo groups and 0 or 1 fused benzo rings. A nonexclusivelist containing specific examples of such heterocycles are as follows:

wherein the crossed bond represents that the heterocycle may be attachedat any available position on the ring that it contacts.

Some of the compounds of the present invention are capable of formingsalts with various inorganic and organic acids and bases and such saltsare also within the scope of this invention. Examples of such acidaddition salts include acetate, adipate, ascorbate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,citrate, cyclohexyl sulfamate, ethanesulfonate, fumarate, glutamate,glycolate, hemisulfate, 2-hydroxyethyl-sulfonate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, hydroxymaleate, lactate,malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nitrate,oxalate, pamoate, persulfate, phenylacetate, phosphate, picrate,pivalate, propionate, quinate, salicylate, stearate, succinate,sulfamate, sulfanilate, sulfate, tartrate, tosylate(p-toluenesulfonate), and undecanoate. Base salts include ammoniumsalts, alkali metal salts such as sodium, lithium and potassium salts,alkaline earth metal salts such as aluminum, calcium and magnesiumsalts, salts with organic bases such as dicyclohexylamine salts,N-methyl-D-glucamine, and salts with amino acids such as arginine,lysine, ornithine, and so forth. Also, basic nitrogen-containing groupsmay be quaternized with such agents as: lower alkyl halides, such asmethyl, ethyl, propyl, and butyl halides; dialkyl sulfates likedimethyl, diethyl, dibutyl; diamyl sulfates; long chain halides such asdecyl, lauryl, myristyl and stearyl halides; aralkyl halides like benzylbromide and others. Non-toxic physiologically-acceptable salts arepreferred, although other salts are also useful, such as in isolating orpurifying the product.

The salts may be formed by conventional means, such as by reacting thefree base form of the product with one or more equivalents of theappropriate acid in a solvent or medium in which the salt is insoluble,or in a solvent such as water, which is removed in vacuo or by freezedrying or by exchanging the anions of an existing salt for another anionon a suitable ion-exchange resin.

Estrogen Receptor Binding Measurements

Abbreviated Procedure for Fluorescence Polarization Estrogen Receptor(ERFP) Binding Assay

A homogeneous mix-and-measure estrogen receptor (ER) binding assay whichutilizes fluorescence polarization (FP) technology is used to identifycompounds with affinity for the estrogen receptor. Purchased fromPanVera (Madison, Wis.), assay reagents include purified humanrecombinant ERα, human recombinant ERβ, ES2 screening buffer (100 mMpotassium phosphate, pH 7.4, 100 μg/mL bovine gamma globulin), andFluormone™ ES2. Fluormone™ ES2, whose formulation is proprietary toPanVera, is a fluorescein-tagged, estrogen-like molecule which exhibitsapproximately equal affinity for ERα and ERβ.

For competition binding experiments, dilutions of test compounds areprepared at 2× the final assay concentration in 0.2% DMSO in ES2Screening buffer on TECAN Genosys, and 25 μL compound/well is dispensedinto black Costar ½ volume 96-well plates. Dependent upon a lot specificK_(d) determination, 10-40 nM ERα or 10-40 nM ERβ and 1 nM Fluormone ES2are then added to these plates in a final assay volume of 50 μL/well.Plates are gently shaken for at least 5 minutes to mix and incubated forat least 1 hr 45 minutes to achieve equilibrium. (Reaction mixtures arestable for up to 5 hours). After centrifugation to remove air bubbles,plates are read on an LJL Analyst or Acquest equipped with Criterionsoftware at the following settings: Fluorescence Polarization Mode;Static Polarizer on Excitation Side; Dynamic Polarizer on Emission Side;Excitation λ=485±10 nm; Emission λ=520±12.5 nm.

Polarized fluorescence intensity values are collected and subsequentlyconverted electronically to millipolarization (mp) values. Followingdata reduction and normalization with Excel and/or Prism software, %Ctrl values at the various test concentrations are used to obtain IC₅₀values via non-linear regression analysis of a four-parameter logisticequation.

Because ligand depletion is a consideration in this assay (˜40-60% inputES2 is bound in the assay), IC₅₀ values are converted to K_(i) valuesthrough application of the Kenakin formula, as outlined in the referencebelow, rather than via the more routinely-used Cheng-Prusoff formula.

Reference: Bolger et al., Rapid Screening of Environmental Chemicals forEstrogen Receptor Binding Capacity, Environmental Health Pespectives:106(1998), 1-7.

Cell-Based Assay for ER Transcriptional Activity:

ERs are ligand-dependent transcription factors that bind the promoterregions of genes at a consensus DNA sequence called the estrogenresponsive element (ERE). The ER agonist or antagonist activity of adrug was determined by measuring the amount of reporter enzyme activityexpressed from a plasmid under the control of an estrogen-responsiveelement when cells transiently transfected with ER and the reporterplasmid were exposed to drug. These experiments were conducted accordingto the following methods.

Plasmids:

Estrogen Receptors alpha (αER, Gen Bank accession #M12674), and beta(βER, Gen Bank #X99101 were cloned into the expression vector pSG5(Stratagene). A trimer of the vitellogenin-gene estrogen responseelement (vitERE) was synthesized as an oligonucleotide and attached to abeta-globin basal promoter in a construct named pERE3gal. This responseelement and promoter were removed from pERE3gal by digestion with theendonucleases SpeI (filled with Klenow fragment) and HindIII. Thisblunt/Hind III fragment was cloned into the β-galactosidase (β-gal)enhancer reporter plasmid (pBGALenh, Stratagene). αER and βER plasmidswere purified using a the Endo Free Maxi Kit (Qiagen), and the DNAconcentration and purity (A260/280 ratio) were determinedspectrophotometrically (Pharmacia). Only DNA with A260/280 ratio of 1.8and a concentration of >1 ug/uL was used for transfections.

Vitellogenin Response Element Sequence: CTAGT CTCGAG AGGTCACTGTGACCT

AGGTCACTGTGACCTAGATCTAGGTCACTGTGACCT AC          =Spel overhang         =Xhol site          =AfIII overhang          =ERE consensus         =

Cells:

All Transfections are performed in 293 cells (Human Embryonic Kidneycells ATCC #CRL-1573). Cells are grown in DMEM supplemented with 10%FBS, glutamine, sodium pyruvate and penicilin/streptomycin. Cells aregrown to 70% confluency and split 1:4.

Transfection:

1. 293 cells are split the night before onto collagen I-coated 150 mmtissue-culture plates (Biocoat, Becton Dickinson #354551) at a densityof 60-70% in DMEM Mediatech 17-205-CV) 10% charcoal-stripped FBS(biocell #6201-31). Approximately 1×10⁷ cells/plate will yield 70%confluency.

2. The next morning, 1 hour prior to transfection, the media is changedto fresh DMEM 10% FBS stripped and supplements.

3. Transfections are performed using the Profection Kit (Promega#E1200). This kit is based on the calcium-phosphate-mediatedtransfection technique. Reagents are added in sterile polystyrene tubesin the following order:

Solution A

-   -   15 μg αER or βER    -   45 μg Reporter (pBGALenh or ERE3)    -   1.5 mL Sterile Water    -   186 μL CaCl₂    -   Mix gently

Solution B

-   -   1.5 mL 2× Hank's Buffered Salt Solution

4. Using a vortex set on low, add solution A to solution B dropwise. Theresulting solution should become milky in color. It is important toachieve thorough mixing. The solution is allowed to settle for 30minutes, then vortexed before adding the solution to cells.

5. Add the mixture to 150 mm plates dropwise. Mix well by rocking platesback and forth and side to side gently. After an hour, a very fineprecipitate should be seen floating on and above cells under 20×magnification. If this precipitate is not observed, the transfectionwill not be effective. Incubate the cells for 12 hours.

Receptor Stimulation:

1. The day after transfection, cells are washed 2× with calcium- andmagnesium-free Mg free PBS containing 1 mM EGTA (pH 7.6). Cells aretrypsinized for 2 min with 3 mL of trypsin-EDTA. Trypsin is neutralizedwith DMEM 10% FCS. Cells are pelleted at 1000×g for 5 min. The cellpellet is then resuspended in 5 mL DMEM plus 2% phenol-red-free FCSsupplemented with glutamine, pyruvate, and Penn/Strep.

2. 50 μl of the resulting cell suspension is plated into each well of96-well tissue culture dishes (Biocoat B&D #354407) using amulti-channel pipettor. The dishes have been previously loaded with 50μL of DMSO-solubilized test compounds at twice the test concentration inDMEM. Data reported are either n=4 wells (single poke) and n=2 wells(9-point concentration-response curves).

3. Cells are incubated overnight at 37° C. in the selected compounds.

Reporter Assay:

1. After 24 h, 100 μL of 7% CPRG (Roche 0884308) cocktail is added toeach well in 1× Z-buffer, the plate is shaken gently at 37° C. for 3 h.CPRG turns bright red as it is cleaved by β-galactosidase.

2. Absorbance measurments (570 nm) were obtained using a plate reader(Molecular Devices).

3. Data is compiled and analyzed using MS Excel. 10 × Z Buffer SodiumPhosphate (dibasic) 1.7 g 600 mM Sodium Phosphate (monobasic) 0.96 g 400mM Potassium Chloride 149 mg 100 mM Magnesium Sulfate 0.2 mL of 1 molarstock 100 mM BME 0.78 mL 500 mM Bring Final Volume to 20 mL withDe-Ionized Water

7% CPRG COCKTAIL

For 50 mLs:

-   -   add 3.5 mL of 50 ml of CPRG    -   add 3.5 mL of 10× Z Buffer    -   add 1 mL of 10% SDS    -   bring to 50 mL with DI water        Typical Results:

Absorbance values illustrating typical concentration-response curvesobtained for the ER agonist 17-β-estradiol (E) and the ER antagonistICI182,780 (A) are plotted below for cells transfected with either αERor βER.

Administration and Use

Compounds of the present invention are shown to have high selectivityfor ER-β over ER-α, and may possess agonist activity on ER-β withoutundesired uterine effects. Thus, these compounds, and compositionscontaining them, maybe used as therapeutic agents in the treatment ofvarious CNS diseases related to ER-β, such as, for example, Alzheimer'sdisease.

The present invention also provides compositions comprising an effectiveamount of compounds of the present invention, including the nontoxicaddition salts, amides and esters thereof, which may, serve to providethe above-recited therapeutic benefits. Such compositions may also beprovided together with physiologically-tolerable liquid, gel or soliddiluents, adjuvants and excipients. The compounds of the presentinvention may also be combined with other compounds known to be used astherapeutic agents for the above or other indications.

These compounds and compositions may be administered by qualified healthcare professionals to humans in a manner similar to other therapeuticagents and, additionally, to other mammals for veterinary use, such aswith domestic animals. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectionmay also be prepared. The preparation may also be emulsified. The activeingredient is often mixed with diluents or excipients which arephysiologically tolerable and compatible with the active ingredient.Suitable diluents and excipients are, for example, water, saline,dextrose, glycerol, or the like, and combinations thereof. In addition,if desired the compositions may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, stabilizing orpH-buffering agents, and the like.

The compositions are conventionally administered parenterally, byinjection, for example, either subcutaneously or intravenously.Additional formulations which are suitable for other modes ofadministration include suppositories, intranasal aerosols, and, in somecases, oral formulations. For suppositories, traditional binders andexcipients may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient. Oral formulations include such normally employedexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, magnesiumcarbonate, and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained-release formulations,or powders.

In addition to the compounds of the present invention that display ER-βactivity, compounds of the present invention can also be employed asintermediates in the synthesis of such useful compounds.

Synthesis

Compounds within the scope of the present invention may be synthesizedchemically by means well known in the art. The following Examples aremeant to show general synthetic schemes, which may be used to producemany different variations by employing various commercially availablestarting materials. These Examples are meant only as guides on how tomake some compounds within the scope of the invention, and should not beinterpreted as limiting the scope of the invention.

EXAMPLES

Example R¹ R^(2a) R^(2b) R^(2c) 1 H H OH H 2 H Cl OH H 3 H CH₃ OH H 4 HOCH₃ H NO2 5 H NO₂ OH H 6 phenyl H OH H 7 2,4-dimethylphenyl H OH H 84-methylsulfanylphenyl H OH H 9 4-trifluoromethylphenyl H OH H 102,4-dichlorophenyl H OH H 11 4-ethylphenyl H OH H 12 o-tolyl H OH H 13 HCl Cl H 14 methyl H OH H 15 eethyl H OH H 16 benzyl H OH H 17 methyl H HOH 18 methyl CH₃ H OH 19 ethyl H OH Cl 20 phenyl H CH₃ OH 21 3-furyl HCH₃ OH 22 2-thiophene H CH₃ OH

Example R⁶ R⁷ 23 OH H 24 H OH Ex- am- Synthetic HPLC FP β-ER FP α-ER FPple Method (method) MS K_(i) (nM) K_(i) (nM) Selectivity 1 A, B 0.46 (A)242.0 375 1000 2.7 2 A, B 4.60 (C) 276.1 18 48 2.7 3 A, B 0.61 (A) 256.08 60 7.4 4 A, B 2.05 (B) 301.2 459 655 1.4 5 A, B 1.88 (B) 2287.3 13 6224.8 6 C, A, B 1.58 (A) 318.4 10 10 0.9 7 D, E, B 1.83 (A) 346.4 35 621.8 8 D, E, B 1.78 (A) 364.4 39 18 0.5 9 D, E, B 1.58 (A) 386.4 4.9 81.6 10 D, E, B 1.97 (A) 386.3, 7 11 1.6 388.3 11 D, E, B 1.85 (A) 346.29 10 1.1 12 D, E, B 1.67 (A) 332.4 8 16 2.2 13 A, B 2.95 (A) 294.3, 3255 1.8 296.3 14 F, G, H, B 1.57 (D) 256.4 45 307 6.8 15 G, H, B 1.74 (D)270.4 18 59 3.3 16 G, H, B 2.03 (A) 332.4 18 15 0.8 17 F, G, H, B 1.44(D) 256.2 34 110 3.3 18 F, G, H, B 1.28 (D) 270.3 4.5 9 2.1 19 F, G, H,B 1.80 (D) 304.2, 24.4 72 3.0 306.2 20 F, G, H, B 1.89 (D) 332.5 1.2 3.02.5 21 F, G, H, B 1.71 (D) 322.5 3.8 22 6.0 22 F, G, H, B 2.05 (D)3338.5 3.5 12 3.5 23 I, B 270.0 229 660 2.9 24 I, B 270.2 215 660 3.1 25J, K 228 55 190 3.5 26 J, K, L 256 160 200 1.3 27 J, K, L 304 190 1700.9 ERE ERE β-ER α-ER EC₅₀ ERE β-ER EC₅₀ ERE α-ER ERE Example (nM) Max(nM) Max Selectivity 1 2 3 35.7 103 28.2 93 0.8 4 5 6 7.6 105 0.6 1000.1 7 8 9 10 44 95 11 12 71 113 13 69 89 14 143 44 1000 42 7.0 15 23 511000 37 43 16 17 49 72 2.5 87 0.1 18 6.1 117 24.6 85 4.0 19 209 41 16.866 0.1 20 0.4 85 1.2 75 3.3 21 0.7 106 0.8 77 1.1 22 23 24 25 161 77 19260 1.2 26 79 85 73 102 0.9 27 34 90 6.8 54 0.2HPLC conditions used:HPLC Method A: This method was used unless otherwise stated. 50 × 2.1mmm, Zorbax Stablebond C₈ colimn; flow rate 1.4 mL/min, linear gradientfrom 15% B to 90% B over 4 min; A = water, 0.05% TFA; B = 90% CH₃CN, 10%water, 0.05% TFA; UV detection at 215 nm.HPLC Method B:The same method as for A, except UV detection 254 nm.HPLC Method C: 75 × 4.60 mm, 3 mm, C₁₈ Phenomenec-Luma column; flow rate1.0 mL/min, linear gradient from 20% B to 80% B over 10 min; A = water,0.1% TFA; B = CH₃CN, 0.1% TFA; UV detection at 210 and 254 nm.HPLC Method D: 50 × 2.1 mm, Zorbax Stablebond C₈ column; flow rate 1.4mL/min, linear gradient from 5% B to 90% B over 4 min; A = water, 0.055%TFA; B = 90% CH₃CN, 10% water, 0.05% TFA; DAD detection.CH₃CN: acetonitrileTFA: trifluoroacetic acidDMSO: dimethylsulfoxideCH₂Cl₂: methylene chloride

Example 1 2-(4-Hydroxyphenyl)-1,2,3,4-tetrahydroisoquinoline-6-ol 1)Synthetic Method A: Synthesis of6-methoxy-2-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline

A solution of 6-methoxy-1,2,3,4-tetrahydroisoquinoline [1] (0.130 g) intoluene (1.75 mL) was added to sodium t-butoxide (0.092 g) in a 5 mLreaction vial equipped with a frit. A suspension oftris(dibenzylideneacetone) dipalladium (0) (0.022 g) and(R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (0.040 g) in toluene(0.75 mL) was added to the above mixture. The reaction was agitated at80° C. for 18 h, then cooled and the solids were removed by filteringthe reaction through the frit. The filtrate was evaporated and theresulting residue was purified by chromatography on silica gel (eluant:ethylacetate-hexane, gradient from 5:95 to 1:1) to give the titlecompound (0.065 g). MS: 270.1 (MH⁺); TLC R_(f): 0.33 (20% ethyl acetatehexane); ¹H NMR (DMSO-d₆): 7.04 (d, 1H, J=8.4 Hz), 6.97 (d, 2H, J=9.2Hz), 6.86 (d, 2H, J=9.1 Hz), 6.75 (dd, 1H, J=2.6, 8.4 Hz), 6.68 (d, 1H,J=2.5 Hz), 4.24 (s, 2H), 3.79 (s, 3H), 3.78 (s, 3H), 3.43 (t, 2H, J=5.8Hz), 2.96 (t, 2H, J=5.7 Hz).

Reference 1: made according to J. S. Buck, J. Am. Chem. Soc.; 1934; 56;1769.

2) Synthetic Method B: Synthesis of2-(4-hydroxyphenyl)-1,2,3,4-tetrahydroisoquinoline-6-ol

A 1.0 M solution of boron tribromide in CH₂Cl₂ (0.54 mL) was addeddropwise to a cooled (−15° C.) solution of6-methoxy-2-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline (0.036 g)in CH₂Cl₂ (1.4 mL). After 30 min the reaction was warmed to roomtemperature. After 2 h the reaction was diluted with CH₂Cl₂ (30 mL) andwashed successively with saturated aqueous sodium bicarbonate (2×20 mL)and saturated aqueous sodium chloride (1×15 mL). The organic extract wasdried and evaporated. The resulting residue was purified bychromatography on silica gel (eluant; a gradient of 0 to 20% methanol indichloromethane) to give the title compound (0.026 g); MS: 242.0 (MH⁺);HPLC t_(R): 0.46 min; ¹H NMR (DMSO-d₆): 9.14 (s, 1H), 8.79 (s, 1H), 6.94(d, 1H, J=6 Hz), 6.85 (d, 2H, J=6.65 (d, 2H J=9 Hz), 6.56 (d, 1H, J=6Hz), 6.52 (s, 1H), 4.06.(s, 2H), 3.28 (t, 2H, J=6 Hz), 2.78 (t, 2H, J=6Hz).

Example 22-(2-Chloro-4hydroxyphenyl)-1,2,3,4-tetrahydroisoquinoline-6-ol

According to synthetic methods A and B, from 4-bromo-3-chloroanisole(0.150 g) was obtained2-(2-chloro-4-methoxyphenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline(0.057 g) after purification by chromatography on silica gel (eluant:ethyl acetate-hexane, gradient from 5:95 to 1:1); MS: 304.2 (100%)(MH⁺), 306.2 (40%) (MH⁺); TLC R_(f): 0.47 (20% ethyl acetate:hexane);and 2-(2-chloro-4-hydroxyphenyl)-1,2,3,4-tetrahydroisoquinoline-6-ol(0.044 g) was obtained after purification by chromatography on silicagel (eluant: a gradient of 0 to 4% methanol in dichloromethane); MS:276.1 (MH⁺); HPLC t_(R): 4.60 min (method C); ¹H NMR (MeOD-d₄): 7.06 (d,1H, J=8.7 Hz), 6.90 (d, 1H, J=7.8 Hz), 6.85 (d, 1H, J=2.7 Hz), 6.69 (dd,1H, J=8.9, 2.9 Hz), 6.60 (d, 1H, J=2.7 Hz), 6.57 (s, 1H), 4.01 (s, 2H),3.19 (t, 2H, J=5.7 Hz), 2.90 (t, 2H, J=5.7 Hz).

Example 32-(4-Hydroxy-2-methylphenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

According to synthetic methods A and B, from 2-bromo-5-methoxytoluene(0.15 mL) was obtained6-methoxy-2-(4-methoxy-2-methylphenyl)-1,2,3,4-tetrahydroisoquinoline(0.188 g) after purification by chromatography on silica gel (eluant:ethyl acetate-hexane, gradient from 5:95 to 1:1); MS: 284.1 (MH⁺); TLCR_(f)=0.64 (20% ethyl acetate:hexane); and using the compound isolatedabove (0.094 g), the title compound (0.061 g) was obtained afterpurification by silica gel chromatography (eluant: a gradient from 0 to20% methanol in CH₂Cl₂); MS: 256.0 (MH⁺; HPLC t_(R): 0.61 min; ¹H NMR(DMSO-d₆): 9.12 (s, 1H), 8.97 (s, 1H), 6.93 (d, 1H, J=8.4 Hz), 6.87 (d,1H, J=8.7 Hz), 6.53-6.60 (m, 4H), 3.81 (s, 2H), 2.95-2.97 (m, 2H),2.81-2.82 (m, 2H).

Example 4 2-(2-Methoxy-5-nitrophenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

According to synthetic method A, from 2-bromo-4-nitro-anisole (0.222 g)was obtained6-methoxy-2-(2-methoxy-5-nitrophenyl)-1,2,3,4-tetrahydroisoquinoline(0.068 g) after purification by chromatography on silica gel (eluant:ethyl acetate-hexane, gradient from 5:95 to 1:1); MS: 315.1 (MH⁺); TLCR_(f): 0.28 (20% ethyl acetate; hexane).2-(2-Methoxy-5-nitrophenyl)-1,2,3,4tetrahydroisoquinolin-6-ol (0.033 g)was obtained when the above isolated compound was treated with 1.2equivalents of 1.0 M boron tribromide according to synthetic method Band after purification by silica gel chromatography (eluant: a gradientfrom 0 to 7% methanol in CH₂Cl₂); MS: 301.2 (MH⁺); HPLC t_(R): 2.05 min(method B); ¹H NMR (DMSO-d₆): 9.14 (s, 1H), 7.92 (dd, 1H, J=2.7, 9.0Hz), 7.72 (d, 1H, J=2.7 Hz), 7.17 (d, 1H, J=9.0 Hz), 6.97 (d, 1H, J=8.1Hz), 6.59 (dd, 1H, J=2.3, 8.1 Hz), 6.55 (s, 1H), 4.15 (s, 2H), 3.96 (s,3H), 3.33 (t, 2H, J=5.6 Hz), 2.82 (t, 2H, J=5.5 Hz).

Example 5 2-(4-Hydroxy-2-nitrophenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

According to synthetic method A, from 4-bromo-3-nitroanisole (0.222 g)was obtained6-methoxy-2-(4-methoxy-2-nitrophenyl)-1,2,3,4-tetrahydroisoquinoline(0.057 g) after purification by chromatography on silica gel (eluant:ethyl acetate-hexane, gradient from 5:95 to 1:1); MS: 315.1 (MH⁺); TLCR_(f): 0.32 (20% ethyl acetate hexane). According to synthetic method B,from the above isolated compound the title compound (0.008 g) wasobtained after purification by chromatography on silica gel (eluant: agradient of 0 to 7% methanol in dichloromethane); MS: 287.3 (MH⁺; HPLCt_(R): 1.88 min (method B); ¹H NMR (DMSO-d₆+TFA-d): 7.46 (d, 1H, J=9.0Hz), 7.24 (d, 1H, J=2.7 Hz), 7.11 (dd, 1H, J=6.0, 3.0 Hz), 6.92 (d, 1H,J=8.1 Hz), 6.60-6.64 (m, 2H), 4.14 (s, 2H), 3.26 (t, 2H, J=5.7 Hz), 2.87(t, 2H, J=5.7 Hz).

Example 62-(4Hydroxyphenyl)-1-phenyl-1,2,3,4-tetrahydroisoquinolin-6-ol 1)Synthetic Method C: Synthesis of6-methoxy-1-phenyl-1,2,3,4-tetrahydroisoquinoline

3-Methoxyphenylethylamine (1.5 g) and benzaldehyde (1.0 mL) were reactedtogether in benzene (10 mL) in a modified Dean-Stark apparatus, wherethe side arm contained activated 3 Å molecular sieve beads. The reactionwas vigorously refluxed for 1 h until a uniform solution resulted. Thereaction was cooled and evaporated. The resulting residue was taken upin trifluoroacetic acid (6 mL) and heated to 60° C. for 18 h. Thereaction was cooled, concentrated then partitioned between 1N sodiumhydroxide (70 mL) and diethyl ether (50 mL). The organic layer waswashed with 0.5 N sodium hydroxide (70 mL), dried, concentrated to 20 mLand cooled in an ice-water bath. The crystals that formed were collectedand dried to give the title compound (1.90 g). MS: 240.0 (MH⁺); HPLCt_(R): 1.62 min (method D); ¹H NMR (DMSO-d₆+TFA-d): 7.47-7.49 (m, 3H),7.37-7.39 (m, 2H), 6.90 (broad s, 1H), 6.79 (dd, 1H, J=2.4, 8.7 Hz),6.66 (d, 1H, J=8.7 Hz), 5.75 (s, 1H), 3.77 (s, 3H), 3.39-3.44 (m, 2H),3.17-3.28 (m, 1H), 3.04-3.12 (m, 1H).

2) Synthesis of2-(4-hydroxyphenyl)-1-phenyl-1,2,3,4-tetrahydroisoquinolin-6-ol

According to synthetic method A, from 4-bromoanisole (0.17 mL) wasobtained6-methoxy-2-(4-methoxy-phenyl)-1-phenyl-1,2,3,4-tetrahydroisoquinoline(0.045 g) after purification by chromatography on silica gel (eluant:ethyl acetate-hexane, gradient from 5:95 to 1:1); MS: 346.3 (MH⁺); HPLCt_(R): 2.24 min; TLC R_(f)=0.20 (5% ethyl acetate:hexane). According tosynthetic method B, from the above isolated compound the title compound(0.034 g) was obtained after purification by silica gel chromatography(eluant: a gradient of 0 to 20% methanol in dichloromethane); MS: 318.4(MH⁺); HPLC t_(R): 1.58 min; TLC R_(f): 0.30 (5% methanol; CH₂Cl₂); ¹HNMR (DMSO-d₆): 9.20 (s, 1H), 8.64 (s, 1H), 7.12-7.20 (m, 5H), 6.94 (d,1H, J=7.8 Hz), 6.69 (d, 2H, J=8.8 Hz), 6.54-6.59 (m, 4H), 5.58 (s, 1H),3.35-3.45 (m, 2H), 2.81 (t, 2H, J=5.8 Hz).

Example 71-(2,4-Dimethylphenyl)-2-(4-hydroxyphenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol 1)Synthetic method D: Synthesis of1-(2,4-dimethylphenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline

3-Methoxyphenylethylamine (1.51 g) and 2,4-dimethylbenzaldehyde (1.34 g)were mixed together. Phosphoric acid, 85%, (20 mL) was added then thereaction was warmed to 37° C. for 60 h. The reaction was cooled, pouredinto water (300 mL) then washed with diethyl ether (2×50 mL). Theaqueous layer was diluted to 700 mL with water, made basic (pH=10) bythe slow addition of sodium hydroxide, and then extracted with diethylether (2×150 mL). The organic extracts were dried and evaporated. Theresulting residue was purified by silica gel chromatography (eluant:ethyl acetate-hexane, gradient from 1:1 to 100% ethyl acetate) to givethe title compound (1.458 g). MS: 268.2 (MH⁺); HPLC t_(R): 1.71 min(method D); TLC R_(f): 0.21 (ethyl acetate); ¹H NMR (DMSO-d₆): 6.96 (s,1H), 6.86 (s, 2H), 6.67 (d, 1H, J=2.3 Hz), 6.59 (dd, 1H, J=2.5, 8.5 Hz),6.44 (d, 1H, J=8.5 Hz), 5.07 (s, 1H), 3.69 (s, 3H), 3.03-3.11 (m, 1H),2.82-2.89 (m, 2H), 2.63-2.77 (m, 1H), 2.26 (s, 3H), 2.23 (s, 3H).

2) Synthetic Method E: Synthesis of1-(2,4-dimethylphenyl)-6-methoxy-2-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline

A fine suspension of sodium t-butoxide (0.055 g) and 4-bromoanisole(0.112 g) in toluene (4.66 mL) was added to1-(2,4-dimethylphenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline (0.134g) in a 10 mL reaction vial equipped with a frit. A solution oftris(dibenzylideneacetone) dipalladium (0) (2.52 μmol) and2-dicyclohexylphosphino-2′-((N,N)-dimethylamino)-biphenyl [2] (5.9 mg)in toluene (0.90 mL) was added. The reaction was agitated at 95° C. for34 h, then cooled and the solids were removed by filtering the reactionthrough the frit. The filtrate was evaporated and the resulting residuewas purified by chromatography on silica gel (eluant: a gradient from 0to 20% ethyl acetate in hexane) to give the title compound (0.054 g).MS: 374.5 (MH⁺); HPLC t_(R): 2.40 min; TLC R_(f): 0.61 (20% ethylacetate:hexane).

Reference 2: made according to D. W. Old, J. P. Wolfe, S. L. Buchwald,J. Am. Chem. Soc.; 1998; 120; 9722-9723.

3) Synthesis of1-(2,4-Dimethylphenyl)-2-(4-hydroxyphenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

According to synthetic method B, from1-(2,4-dimethylphenyl)-6-methoxy-2-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline(0.050 g) was obtained the title compound (0.023 g) after silica gelchromatography (eluant: a gradient of 0 to 12% methanol indichloromethane); MS: 346.4 (MH⁺); HPLC t_(R): 1.83 min; ¹H NMR(DMSO-d₆): 9.16 (s, 1H), 8.79 (s, 1H), 6.89 (s, 1H), 6.79-6.84 (m, 2H),6.51-6.66 (m, 7H), 5.54 (s, 1H), 3.15-3.23 (m, 2H), 2.66 (broad s, 2H),2.20 (s, 6H).

Example 82-(4-Hydroxyphenyl)-1-(4-methylsulfanylphenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

According to synthetic method D using 4-(methylthio)benzaldehyde (1.33mL), 3-methoxy-1-(4-methylsulfanylphenyl)-1,2,3,4-tetrahydroisoquinoline(0.441 g) was obtained after silica gel chromatography (eluant: ethylacetate-hexane, gradient from 1:1 to 100% ethyl acetate). MS: 286.2(MH⁺); HPLC t_(R): 1.65 min (method D); TLC R_(f): 0.11 (ethyl acetate);¹H NMR (DMSO-d₆): 7.18 (s, 4H), 6.67 (d, 1H, J=2.2 Hz), 6.59 (dd, 1H,J=2.4, 8.4 Hz), 6.52 (d, 1H, J=8.4 Hz), 4.88 (s, 1H), 3.69 (s, 3H),3.02-3.07 (m, 1H), 2.82-2.88 (m, 2H), 2.66-2.71 (m, 1H), 2.45 (s, 3H).According to synthetic method E using the above isolated compound (0.143g),6-methoxy-2-(4-methoxyphenyl)-1-(4-methylsulfanylphenyl)-1,2,3,4-tetrahydroisoquinoline(0.052 g) was obtained after silica gel chromatography (eluant: agradient from 0 to 20% ethyl acetate in hexane); MS: 392.5 (MH⁺), HPLCt_(R): 2.37 min; TLC R_(f): 0.47 (20% ethyl acetate:hexane). Accordingto synthetic method B using the above isolated compound (0.047 g), thetitle compound (0.034 g) was obtained after silica gel chromatography(eluant: a gradient of 0 to 12% methanol in dichloromethane); MS: 364.4(MH⁺); HPLC t_(R): 1.78 min; ¹H NMR (DMSO-d₆): 9.23 (s, 1H), 8.69 (s,1H), 7.07-7.08 (m, 4H), 6.91 (d, 1H, J=7.5 Hz), 6.69-6.72 (m, 2H),6.56-6.59 (m, 4H), 5.55 (s, 1H), 2.81 (broad s, 2H), 2.50 (broad s, 2H),2.40 (s, 3H).

Example 92-(4-Hydroxyphenyl)-1-(4-trifluoromethylphenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

According to synthetic method D using 4-(trifluoromethyl)benzaldehyde,(1.74 g),6-methoxy-1-(4-trifluoromethylphenyl)-1,2,3,4-tetrahydroisoquinoline(1.208 g) was obtained after silica gel chromatography (eluant: ethylacetate-hexane, gradient from 1:1 to 100% ethyl acetate); MS: 308.2(MH⁺); HPLC t_(R): 1.93 min; TLC R_(f): 0.32 (ethyl acetate). Accordingto synthetic method E using the above isolated compound (0.154 g),6-methoxy-2-(4-methoxyphenyl)-1-(4-trifluoromethylphenyl)-1,2,3,4-tetrahydroisoquinoline(0.063 g) was obtained after silica gel chromatography (eluant: agradient from 0 to 20% ethyl acetate in hexane); MS: 414.4 (MH⁺); HPLCt_(R): 2.78 min; TLC R_(f): 0.41 (20% ethyl acetate: hexane). Accordingto synthetic method B using the above isolated compound, the titlecompound (0.057 g) was obtained after silica gel chromatography (eluant:a gradient of 0 to 8% methanol in dichloromethane); MS: 386.4 (MH⁺);HPLC t_(R): 1.58 min; ¹H NMR (DMSO-d₆): 9.29 (s, 1H), 8.72 (s, 1H), 7.58(d, 2H, J=7.9 Hz), 7.37 (d, 2H, J=7.9 Hz), 6.97 (d, 1H, J=7.9 Hz),6.69-6.73 (m, 2H), 6.56-6.60 (m, 4H), 5.70 (s, 1H), 3.39-3.45 (m, 1H),3.25-3.28 (m, 1H), 2.83 (broad s, 2H).

Example 101-(2,4-Dichlorophenyl)-2-(4-hydroxyphenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

According to synthetic method D using 2,4-dichlorobenzaldehyde (1.75 g),1-(2,4-dichlorophenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline (0.241g) was obtained after silica gel chromatography (eluant; ethylacetate-hexane, gradient from 1:1 to 100% ethyl acetate); MS: 308.3(100%) (MH⁺), 310.4 (60%) (MH⁺); HPLC t_(R): 1.87 min; TLC R_(f); 0.58(ethyl acetate-hexane 1:1). According to synthetic method B using theabove isolated compound (0.154 g),1-(2,4-dichlorophenyl)-6-methoxy-2-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline(0.075 g) was obtained after silica gel chromatography (eluant; agradient from 0 to 20% ethyl acetate in hexane); MS: 414.4 (100%) (MH⁺),416.4 (50%) (MH⁺); HPLC t_(R): 2.95 min; TLC R_(f): 0.59 (20% ethylacetate:hexane). According to synthetic method B using the aboveisolated compound, the title compound (0.062 g) was obtained aftersilica gel chromatography (eluant: a gradient of 0 to 8% methanol indichloromethane); MS: 386.3 (100%), 388.3 (60%) (MH⁺); HPLC t_(R): 1.97min; ¹H NMR (DMSO-d₆): 9.30 (s, 1H), 8.88 (s, 1H), 7.52 (s, 1H), 7.26(d, 1H, J=8.1 Hz), 7.08 (d, 1H, J=8.4 Hz), 6.82 (d, 2H, J=8.4 Hz), 6.70(d, 1H, J=8.2 Hz), 6.52-6.59 (m, 4H), 5.78 (s, 1H), 3.27-3.35 (m, 2H),2.87-2.92 (m, 1H), 2.71-2.76 (m, 1H).

Example 111-(4-Ethylphenyl)-2-(4-hydroxyphenyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

According to synthetic method D using 4-ethylbenzaldehyde (1.37 mL),1-(4-ethylphenyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline (0.977 g) wasobtained after silica gel chromatography (eluant: ethyl acetate-hexane,gradient from 1:1 to 100% ethyl acetate); MS: 268.3 (MH⁺); HPLC t_(R):1.80 min (method D); TLC R_(f): 0.15 (ethyl acetate). According tosynthetic method E using the above isolated compound (0.134 g),1-(4-ethylphenyl)-6-methoxy-2-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquinoline(0.055 g) was obtained after silica gel chromatography (eluant: agradient from 0 to 20% ethyl acetate in hexane); MS: 374.5 (MH⁺); HPLCt_(R): 2.45 min; TLC R_(f), 0.57 (20% ethyl acetate:hexane). Accordingto synthetic method B using the above isolated compound, the titlecompound (0.045 g) was obtained after silica gel chromatography (eluant:a gradient of 0 to 12% methanol in dichloromethane); MS: 346.2 ; (MH⁺);HPLC t_(R): 1.85 min.

Example 122-(4-Hydroxyphenyl)-1-o-tolyl-1,2,3,4-tetrahydroisoquinolin-6-ol

According to synthetic method D using o-toluadlehyde (1.16 mL),6-methoxy-1-o-tolyl-1,2,3,4-tetrahydroisoquinoline (1.072 g) wasobtained after silica gel chromatography (eluant: ethyl acetate-hexane,gradient from 1:1 to 100% ethyl acetate); MS: 254.3 (MH⁺); HPLC t_(R):1.67 min; TLC R_(f): 0.28 (ethyl acetate-hexane 1:1). According tosynthetic method E using the above isolated compound (0.127 g),6-methoxy-2-(4-methoxy-phenyl)-1-o-tolyl-1,2,3,4-tetrahydroisoquinoline(0.062 g) was obtained after silica gel chromatography (eluant: agradient from 0 to 20% ethyl acetate in hexane); MS: 360.5 (MH⁺); HPLCt_(R): 2.26 min; TLC R_(f): 0.46 (20% ethyl acetate:hexane). Accordingto synthetic niethod B using the above isolated compound, the titlecompound (0.044 g) was obtained after silica gel chromatography (eluant:a gradient of 0 to 12% methanol in dichlioromethane); MS: 332.4 (MH⁺);HPLC t_(R): 1.67 min.

Example 13 2-(2,4-Dichloro-phenyl)-1,2,3,4-tetrahydro-isoquinolin-6-ol

According to synthetic methods A and B, from1-bromo-2,4-dichloro-benzene (0.25 g) was obtained2-(2,4-dichloro-phenyl)-6-methoxy-1,2,3,4-tetrahydro-isoquinoline (0.092g) after purification by chromatography on silica gel (eluant: ethylacetate-hexane, gradient from 5:95 to 1:1); MS: 306.3, 308.3, 310.3(MH⁺); HPLC t_(R): 1.67 min; TLC R_(f)=0.72 (20% ethyl acetate:hexane);and using the compound isolated above (0.092 g), the title compound(0.034 g) was obtained after purification by silica gel chromatography(eluant: a gradient from 0 to 5% methanol in CH₂Cl₂); MS: 294.3, 296.3(MH⁺); HPLC t_(R): 2.95 min; TLC R_(f)=0.53 (5% methanol:methylenechloride).

Example 142-(4-Hydroxy-phenyl)-1-methyl-1,2,3,4-tetrahydro-isoquinolin-6-ol 1)Synthetic Method F: Synthesis of(4-methoxy-phenyl)-[2-(3-methoxy-phenyl)-ethyl]-amine

A solution of (3-methoxy-phenyl)-acetyl chloride (5.0 g) in ethylacetate (85 mL) was added to a solution of 4methoxy-phenylamine (3.33 g)in ethyl acetate (50 mL). After 18 h the reaction was poured into water(150 mL). The organic extract was dried over sodium sulfate, filteredthrough celite and concentrated. The resulting residue was dissolved intetrahydrofurane (100 mL) then lithium aluminum hydride (5.2 g) wasadded in small portions over 30 minutes. After 18 h the reaction wasslowly poured onto 200 mL of ice. The mixture was filtered through a padof celite and the collected filtrate was extracted with ethyl acetate(300 mL). The organic extract was dried over sodium sulfate, filteredthrough celite and concentrated to give the title compound. MS: 258.3(MH⁺); HPLC t_(R): 1.98 min.

2) Synthetic Method G: Synthesis ofN-(4-methoxy-phenyl)-N-[2-(3-methoxy-phenyl)-ethyl]-acetamide

(4-Methoxy-phenyl)-[2-(3-methoxy-phenyl)-ethyl]-amine (3.87 mmol) andacetyl chloride (4.26 mmol) were reacted ethyl acetate (50 mL). After 18hours, the reaction was poured into ice (150 mL) and the organic extractwas dried over sodium sulfate, filtered through celite and concentrated.The title compound (0.60 g) was obtained after silica gel chromatography(eluant: gradient from 10% to 40% ethyl acetate-hexane); MS: 300.4(MH⁺); HPLC t_(R): 2.65 min; TLC R_(f): 0.19 (ethyl acetate-hexane 2:3).

3) Synthetic Method H: Synthesis of6-methoxy-2-(4-methoxy-phenyl)-1-methyl-1,2,3,4-tetrahydro-isoquinoline

N-(4-Methoxy-phenyl)-N-[2-(3-methoxyphenyl)-ethyl]-acetamide (0.60 g)was heated to 80° C. in phosphorus oxychloride (8.4 mL) for 24 h. Thereaction was cooled then poured slowly onto ice (150 mL). Potassiumiodide (0.65 g) was added. After 30 min, the mixture was extracted withmethylene chloride (2×50 mL). The organic extracts were dried oversodium sulfate, filtered through celite and concentrated. The resultingresidue was dissolved in methanol (21 mL) and sodium borohydride (0.23g) was added slowly in small portions. After 18 h the solvent wasremoved under reduced pressure. The residue was partitioned betweenwater (100 mL) and ethyl acetate (50 mL) and the aqueous layer wasextracted with ethyl acetate (25 mL). The combined organic extracts weredried over sodium sulfate, filtered through celite and concentratedgiving the title compound MS: 284.3 (MH⁺); HPLC t_(R); 2.11 min (methodD).

According to synthetic method B,2-(4-hydroxy-phenyl)-1-methyl-1,2,3,4-tetrahydro-isoquinolin-6-ol wasobtained (0.085 g) after purification by chromatography on silica gel(eluant: a gradient of 0 to 4% methanol in dichloromethane); MS: 256.4(MH⁺); HPLC t_(R): 1.57 min (method D); TLC R_(f): 0.36 (5%methanol:methylene chloride).

Example 151-Ethyl-2-(4-hydroxy-phenyl)-1,2,3,4-tetrahydro-isoquinolin-6-ol

According to synthetic method G,N-(4-methoxy-phenyl)-N-[2-(3-methoxy-phenyl)-ethyl]-propionamide (0.573g) was obtained from propionyl chloride (1.2 g). MS: 314.4 (MH⁺); HPLCt_(R): 2.78 min; TLC R_(f): 0.41 (ethyl acetate-hexane 2:3). Accordingto synthetic method H,1-ethyl-6-methoxy-2-(4-methoxy-phenyl)-1,2,3,4-tetrahydro-isoquinolinewas obtained. MS: 298.4 (MH⁺); HPLC t_(R): 2.26 min (method D).According to method B, the title compound was obtained (0.053 g) afterpurification by chromatography on silica gel (eluant: a gradient of 0 to4% methanol in dichloromethane); MS: 270.4 (MH⁺); HPLC t_(R): 1.74 min(method D); TLC R_(f): 0.41 (5% methanol:methylene chloride).

Example 161-Benzyl-2-(4-hydroxy-phenyl)-1,2,3,4-tetrahydro-isoquinolin-6-ol

According to synthetic method G,N-(4-methoxy-phenyl)-N-[2-(3-methroxy-phenyl)-ethyl]-2-phenyl-acetamide(1.19 g) was obtained from phenyl-acetyl chloride (1.14 g). MS: 376.4(MH⁺); HPLC t_(R): 3.03 min; TLC R_(f): 0.48 (ethyl acetate-hexane 2:3).According to synthetic method H,1-benzyl-6-methoxy-2-(4-methoxy-phenyl)-1,2,3,4-tetrahydro-isoquinolinewas obtained. MS: 360.5 (MH⁺); HPLC t_(R): 2.69 min. According to methodB, the title compound was obtained (0.609 g) after purification bychromatography on silica gel (eluant: a gradient of 0 to 10% methanol indichloromethane); MS: 332.4 (MH⁺); HPLC t_(R): 2.03 min; TLC R_(f): 0.6(5% methanol:methylene chloride).

Example 172-(3-Hydroxy-phenyl)-1-methyl-1,2,3,4-tetrahydro-isoquinolin-6-ol

According to synthetic method F,(3-methoxy-phenyl)-[2-(3-methoxy-phenyl)-ethyl]-amine was obtained from3-methoxy-phenylamine (3.33 g). MS: 258.3 (MH⁺); HPLC t_(R): 2.21 min.According to synthetic method G,N-(3-methoxy-phenyl)-N-[2-(3-methoxy-phenyl)-ethyl]-acetamide (0.73 g)was obtained from acetyl chloride (0.53 g). MS: 300.3 (MH⁺); HPLC t_(R):2.51 min (method D); TLC R_(f): 0.36 (ethyl acetate:hexane-1:1).According to synthetic methods H and B, the title compound (0.184 g) wasobtained after purification by chromatography on silica gel (eluant: agradient of 0 to 5% methanol in dichloromethane); MS: 256.2 (MH⁺); HPLCt_(R): 1.44 min (method D); TLC R_(f): 0.10 (2% methanol:methylenechloride).

Example 182-(5-Hydroxy-2-methyl-phenyl)-1-methyl-1,2,3,4-tetrahydro-isoquinolin-6-ol

According to synthetic methods F and G,N-(5-methoxy-2-methyl-phenyl)-N-[2-(3-methoxy-phenyl)-ethyl]-acetamide(0.75 g) was obtained from (3-methoxy-phenyl)-acetyl chloride (4.0 g)and 5-methoxy-2-methyl-phenylamine (2.97 g) followed by reaction withacetyl chloride (1.5 eq). MS: 314.3 (MH⁺); HPLC t_(R): 2.63 min (methodD); TLC R_(f): 0.16 (ethyl acetate:hexane-3:7). According to syntheticmethods H and B, the title compound (0.026 g) was obtained afterpurification by chromatography on silica gel (eluant: a gradient of 0 to50% ethyl acetate in hexane); MS: 270.3 (MH⁺); HPLC t_(R): 1.28 min(method D); TLC R_(f): 0.36 (ethyl acetate:hexane 2:3).

Example 192-(3-Chloro-4-hydroxy-phenyl)-1-ethyl-1,2,3,4-tetrahydro-isoquinolin-6-ol

According to synthetic methods F and G,N-(3-chloro-4-methoxy-phenyl)-N-[2-(3-methoxy-phenyl)-ethyl]-propionamide(0.753 g) was obtained from (3-methoxy-phenyl)-acetyl chloride (1.0 eq)and 3-chloro-4-methoxy-phenylamine (1.0 eq) followed by reaction withpropionyl chloride (1.5 eq). MS: 348.4 (MH⁺); HPLC t_(R): 2.72 mm(method D); TLC R_(f): 0.48 (ethyl acetate:hexane-1:1). According tosynthetic methods H and B, the title compound (0.25 g) was obtainedafter purification by chromatography on silica gel (eluant: a gradientof 0 to 5% methanol in methylene chloride); MS: 304.2, 306.2 (MH⁺); HPLCt_(R): 1.80 min (method D); TLC R_(f): 0.16 (2% methanol:methylenechloride).

Example 202-(3-Hydroxy-4-methyl-phenyl)-1-phenyl-1,2,3,4-tetrahydro-isoquinolin-6-ol

According to synthetic methods F and G,N-(3-methoxy-4-methyl-phenyl)-N-[2-(3-methoxy-phenyl)-ethyl]-benzamide(0.783 g) was obtained from (3-methoxy-phenyl)-acetyl chloride (1.0 eq)and 3-methoxy-4-methyl-phenylamine (1.0 eq) followed by reaction withbenzoyl chloride (1.5 eq). MS: 376.3 (MH⁺); HPLC t_(R): 2.93 min (methodD); TLC R_(f): 0.45 (ethyl acetate:hexane-3:7). According to syntheticmethods H and B, the title compound (0.25 g) was obtained afterpurification by chromatography on silica gel (eluant: a gradient of 0 to50% ethyl acetate in hexane); MS: 332.5 (MH⁺); HPLC t_(R): 1.89 min(method D); TLC R_(f): 0.40 (ethyl acetate:hexane 2:3).

Example 211-Furan-3-yl-2-(3-hydroxy-4-methyl-phenyl)-1,2,3,4-tetrahydro-isoquinolin-6-ol

According to synthetic methods F and G, furan-3-carboxylic acid(3-methoxy-4-methyl-phenyl)-[2-(3-methoxy-phenyl)-ethyl]-amide (0.409 g)was obtained from (3-methoxy-phenyl)-acetyl chloride (1.0 eq) and3-methoxy-4-methyl-phenylamine (1.0 eq) followed by reaction withfuran-3-carbonyl chloride (1.5 eq). MS: 366.3 (MH⁺); HPLC t_(R): 2.86min (method D); TLC R_(f): 0.44 (ethyl acetate:hexane-3:7). According tosynthetic methods H and B, the title compound (0.02 g) was obtainedafter purification by chromatography on silica gel (eluant: a gradientof 0 to 50% ethyl acetate in hexane); MS: 322.5 (MH⁺); HPLC t_(R): 1.71.min (method D); TLC R_(f): 0.48 (ethyl acetate:hexane 2:3).

Example 222-(3-Hydroxy-4-methyl-phenyl)-1-thiophen-2-yl-1,2,3,4-tetrahydro-isoquinolin-6-ol

According to synthetic methods F and G, thiophene-2-carboxylic acid(3-methoxy-4-methyl-phenyl)-[2-(3-methoxy-phenyl)-ethyl]-amide (0.583 g)was obtained from (3-methoxy-phenyl)-acetyl chloride (1.0 eq) and3-methoxy-4methyl-phenylamine (1.0 eq) followed by reaction withthiophene-2-carbonyl chloride (1.5 eq). MS: 382.3 (MH⁺); HPLC t_(R):2.96 min (method D); TLC R_(f): 0.52 (ethyl acetate:hexane-3:7).According to synthetic methods H and B, the title compound (0.04 g) wasobtained after purification by chromatography on silica gel (eluant: agradient of 0 to 50% ethyl acetate in hexane); MS: 338.5 (MH⁺); HPLCt_(R): 2.05 min (method D); TLC R_(f): 0.44 (ethyl acetate:hexane 2:3).

Example 23(6-Hydroxy-3,4-dihydro-1H-isoquinolin-2-yl)-(4-hydroxy-phenyl)-methanone. 1)Synthetic Method I:(6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl)-(4-methoxy-phenyl)-methanone

4-Methoxy-benzoyl chloride (2.35 g) was added dropwise to a solution of6-methoxy-1,2,3,4-tetrahydro-isoquinoline (0.75 g) and triethylamine(1.54 mL) in dichloromethane (40 mL) at 0° C. After 18 h at roomtemperature, the reaction was washed with 1N NaOH (2×50 mL), then water(2×50 mL). The organic extract was dried over sodium sulfate, filteredthrough celite and concentrated. The(6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl)-(4-methoxy-phenyl)-methanone(0.95 g) product was obtained after purification by chromatography onsilica gel (eluant: methylene chloride).

According to synthetic method B, the title compound (0.18 g) wasobtained using(6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl)-(4-methoxy-phenyl)-methanone(0.4 g) and after purification by chromatography on silica gel (eluant:2% methanol:methylene chloride). MS: 270.0 (MH⁺).

Example 24(7-Hydroxy-3,4-dihydro-1H-isoquinolin-2-yl)-(4-hydroxy-phenyl)-methanone

According to synthetic method I,(7-methoxy-3,4-dihydro-1H-isoquinolin-2-yl)-(4-methoxy-phenyl)-methanone(0.79 g) was obtained using 7-methoxy-1,2,3,4tetrahydro-isoquinoline(0.75 g). MS: 298.1 MH⁺. According to synthetic method B, the titlecompound (0.22 g) was obtained using(7-methoxy-3,4-dihydro-1H-isoquinolin-2-yl)-(4-methoxy-phenyl)-methanone(0.44 g) and after purification by chromatography on silica gel (eluant:tetrahydrofuran:methylene chloride 0:10 to 1:9). MS: 270.2 (MH⁺).Isoindolines

Example R⁶ R⁷ R¹ 25 H OH H 26 H OH Et 27 H OH Ph OH H Ph

Example 25 5-Hydroxy-2-(4-hydroxyphenyl)isoindoline 1) Synthesis of5-hydroxy-2-(4-hydroxyphenyl)isoindoline-1,3-dione

A suspension of 4-hydroxyphthalic acid (3.00 g) and 4-aminophenol (1.98g) in glacial acetic acid (15 mL) was heated under nitrogen at 120° C.for 1.5 h. The brown reaction solution was cooled to room temperature,poured into water (200 mL) and allowed to sit undisturbed for 30 min.The precipitate was collected by filtration and washed with water (2×60mL). The solid was dried under high vacuum at 50° C. for 18 h yieldingthe title compound (3.14 g) as a tan solid. ¹H NMR (DMSO-d₆): 10.99 (s,1H), 9.72 (s, 1H), 7.76 (d, 1H, J=7.8 Hz), 7.18 (m, 4M), 6.86 (m, 2H);MS: 256 (MH⁺).

2) Synthesis of 5-benzyloxy-2-(4-benzyloxyphenyl)isoindoline-1,3-dione

To 5-hydroxy-2-(4-hydroxyphenyl)isoindoline-1,3-dione (2.08 g) in DMF(20 mL) was added potassium carbonate (4.62 g) and a solution of benzylbromide (3.07 g) in DMF (3.0 mL) dropwise under nitrogen atmosphere. Thereaction was stirred at room temperature for 2 h then heated to 80° C.for 3 h. The mixture was cooled to room temperature, poured into water(250 mL) and let sit undisturbed for 2.0 h. The white solid wascollected by filtration and washed with water. The solid was dried underhigh vacuum at 50° C. for 18 h yielding the title compound (3.30 g) as awhite solid. ¹H NMR (DMSO-d₆): 7.87 (d, 1H, J=8.4 Hz), 7.48-7.35 (m,12H), 7.32 (d, 2H, J=8.7 Hz), 7.13 (d, 2H, J=8.7 Hz), 5.35 (s, 2H), 5.17(s, 2H); MS: 436 (MH⁺).

3) Synthetic Method J: Synthesis of5-benzyloxy-2-(4-benzyloxyphenyl)isoindoline

To a stirred suspension containing LiAlH₄ (0.133 g) in THF (10 mL) undernitrogen atmosphere was added5-benzyloxy-2-(4-benzyloxyphenyl)isoindoline-1,3-dione (0.435 g) in THF(15 mL) dropwise. The reaction was stirred at room temperature for 30min, poured into cold, saturated ammonium chloride (100 mL) andextracted with ethyl acetate (2×75 mL). The organic layer was washedwith brine and dried over MgSO₄. After evaporation of the solvent, theresidue was dried under high vacuum yielding the title compound (0.4 g)as a brown solid. ¹H NMR (DMSO-d₆): 7.45-7.31 (m, 13H), 6.70 (d, 2H,J=8.5 Hz), 6.51 (d, 2H, J=8.5 Hz), 5.12 (m, 4H), 4.41 (m, 4H); MS: 408(MH⁺)

4) Synthetic Method K: Synthesis of5-hydroxy-2-(4-hydroxyphenyl)isoindoline

5-Benzyloxy-2-(4-benzyloxyphenyl)isoindoline (400 mg) in TFA (12 mL) wasrefluxed under nitrogen atmosphere for 1 h and cooled. The solvents wereremoved under vacuum. The residue was dissolved in ethyl acetate (100mL) and washed with saturated sodium bicarbonate (twice) then brine.Purification by chromatography on silica gel (eluant:dichloromethane—methanol 95:5) gave the title compound (95 mg) as a tansolid. ¹H NMR (DMSO-d₆): 9.35 (s, 1H), 8.53 (s, 1H), 7.14 (d, 1H, J=8.4Hz), 6.75 (s br, 1H), 6.69 (m, 3H), 6.49 (d, 2H, J=8.7 Hz), 4.38 (m,4H); MS: 228 (MH⁺).

Example 26 1-Ethyl-6-hydroxy-2-(4-hydroxyphenyl)isoindoline 1) SyntheticMethod L: Synthesis of5-benzyloxy-2-(4-benzyloxyphenyl)-3-ethyl-3-hydroxyisoindolin-1-one

To a cooled (3° C.) solution containing5-benzyloxy-2-(4-benzyloxyphenyl)-isoindoline-1,3-dione (870 mg) in THF(25 mL) under nitrogen atmosphere was added ethylmagnesium bromide (4mL, 3M solution in THF) dropwise. The mixture was stirred at 3° C. for15 min then allowed to warm to room temperature for 2 h. The mixture wascooled, poured into saturated ammonium chloride and extracted with ethylacetate. The organic layer was washed with brine. Evaporation of thesolvent yielded the title compound (900 mg) as a tan solid. MS: 466(MH⁺).

2) Synthesis of 6-benzyloxy-2-(4-benzyloxyphenyl)-1-ethylisoindoline

According to synthetic method J except that the mixture was stirred atroom temperature for 1 h and at 35° C. for 1 h, from5-benzyloxy-2-(4-benzyloxyphenyl)-3-ethyl-3-hydroxyisoindolin-1-one (0.5g) was obtained 6-benzyloxy-2-(4-benzyloxyphenyl)-1-ethylisoindoline(0.5 g). MS: 436 (MH⁺).

3) Synthesis of 1-ethyl-6-hydroxy-2-(4-hydroxyphenyl)isoindoline

According to synthetic method K, from6-benzyloxy-2-(4-benzyloxyphenyl)-1-ethylisoindoline (435 mg) wasobtained 1-ethyl-6-hydroxy-2-(4-hydroxyphenyl)isoindoline (140 mg). ¹HNMR (D)MSO-d₆): 9.32 (s br, 1H), 8.58 (s br, 1H), 7.31 (d, 1H, J=8.3Hz), 7.15 (d, 1H, J=8.3 Hz), 6.70 (m, 3H), 6.57 (m, 2H), 4.95 (m, 1H),4.55 (m, 1H), 4.31 (m, 1H), 1.98 (m, 1H), 1.74 (m, 1H), 0.56 (m, 3H);MS: 256 (MH⁺).

Example 27 6-Hydroxy-2-(4-hydroxyphenyl)-1-phenylisoindoline and5-hydroxy-2-(4-hydroxyphenyl)-1-phenylisoindoline 1) Synthesis of5-benzyloxy-2-(4-benzyloxyphenyl)-3-hydroxy-3-phenylisoindolin-1-one and6-benzyloxy-2-(4-benzyloxyphenyl)-3-hydroxy-3-phenylisoindolin-1-one

According to synthetic method L, from5-benzyloxy-2-(4-benzyloxyphenyl)-isoindoline-1,3-dione (870 mg) andphenylmagnesium bromide (1M solution in THF, 12 mL) was obtained amixture of5-benzyloxy-2-(4-benzyloxyphenyl)-3-hydroxy-3-phenylisoindolin-1-one and6-benzyloxy-2-(4-benzyloxyphenyl)-3-hydroxy-3-phenylisoindolin-1-one(1.1 g) as a tan foam. MS: 514 (MH⁺).

2) Synthesis of 6-benzyloxy-2-(4-benzyloxyphenyl)-1-phenylisoindolineand 5-benzyloxy-2-(4-benzyloxyphenyl)-1-phenylisoindoline

According to synthetic method J except that the mixture was stirred atroom temperature for 1 h and at 35° C. for 1 h, from5-benzyloxy-2-(4-benzyloxyphenyl)-3-hydroxy-3-phenylisoindolin-1-one(0.53 g) was obtained a mixture of6-benzyloxy-2-(4-benzyloxyphenyl)-1-phenylisoindoline and5-benzyloxy-2-(4-benzyloxyphenyl)-1-phenylisoindoline (0.48 g). MS: 484(MH⁺).

3) Synthesis of 6-hydroxy-2-(4-hydroxyphenyl)-1-phenylisoindoline and5-hydroxy-2-(4-hydroxyphenyl)-1-phenylisoindoline

According to synthetic method K, from the mixture above (480 mg) wasobtained a mixture of 6-hydroxy-2-(4-hydroxyphenyl)-1-phenylisoindolineand 5-hydroxy-2-(4-hydroxyphenyl)-1-phenylisoindoline (85 mg). HPLC(HPLC 2.1×50 mm C₈ 5 μm Zorbax Stablebond column; flow rate 1.4 mL/min,linear gradient from 15% B to 90% B over 4.0 min; A=water, 0.05% TFA;B=90% acetonitrile, 10% water, 0.05% TFA, UV detection at 254 nm andpositive ionization mass spectrometry detection) t_(R): 1.70 min [33%;MS: 304 (MH⁺)] and 1.83 min [66%; MS: 304 (MH⁺)].

1. A compound having the formula:

or a pharmaceutically acceptable salt or hydrolyzable ester thereof,wherein: R¹ is H, C₁₋₈alkyl, phenyl, or a 5- or 6-membered ringheterocycle containing 1, 2 or 3 heteroatoms each independently selectedfrom O, N and S and additionally having 0 or 1 oxo groups and 0 or 1fused benzo rings, wherein the C₁₋₈alkyl, phenyl or heterocycle issubstituted by 0, 1, 2 or 3 substituents selected from —R^(a), —OR^(a),—SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃haloalkyl; R²is C₁₋₈alkyl, phenyl, —C(═O)phenyl, benzyl or a 5- or 6-membered ringheterocycle containing 1, 2 or 3 heteroatoms each independently selectedfrom O, N and S and additionally having 0 or 1 oxo groups and 0 or 1fused benzo rings, wherein the C₁₋₈alkyl, phenyl, —C(═O)phenyl, benzylor heterocycle is substituted by 1, 2 or 3 substituents selected from—OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro and C₁₋₃haloalkyl; and wherein the phenyl, —C(═O)phenyl,benzyl or heterocycle is additionally substituted by 0, 1 or 2substituents selected from C₁₋₆alkyl, phenyl and benzyl; R³ is hydrogen,C₁₋₆alkyl, —(CH₂)_(m)phenyl or —(CH₂)_(m)heterocycle, wherein theheterocycle is a 5- or 6-membered ring heterocycle containing 1, 2 or 3heteroatoms each independently selected from O, N and S and additionallyhaving 0 or 1 oxo groups and 0 or 1 fused benzo rings; R⁴ is H, halogen,C₁₋₆alkyl, C₁₋₆haloalkyl, —(CH₂)_(m)phenyl or —(CH₂)_(m)heterocycle,wherein the heterocycle is a 5- or 6-membered ring heterocyclecontaining 1, 2 or 3 heteroatoms each independently selected from O, Nand S and additionally having 0 or 1 oxo groups and 0 or 1 fused benzorings; R⁵ is —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),—OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl; or R⁵ is C₁₋₃alkyl containing 1 or 2substituents selected from —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),—OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano and nitro; R⁶ is —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a),—CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a),—NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —(═O)R^(a),—S(═O)₂R^(a), halogen, cyano, nitro or C₁₋₃haloalkyl; R⁷ is —R^(a),—OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl; R⁸ is —R^(a), —OR^(a), —SR^(a),—NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano, nitro or C₁₋₃haloalkyl; or R⁸is C₁₋₃alkyl containing 1 or 2 substituents selected from —OR^(a),—SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a), —C(═O)NR^(a)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —C(═O)R^(a),—S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano and nitro; R⁹ is H, C₁₋₅alkylor C₁₋₃haloalkyl; R^(a) is H, C₁₋₆alkyl, phenyl or benzyl; m is 0, 1, 2or 3; and n is 0 or
 1. 2. The compound according to claim 1, wherein R¹is H, phenyl or a 5- or 6-membered ring heterocycle containing 1, 2 or 3heteroatoms each independently selected from O, N and S and additionallyhaving 0 or 1 oxo groups and 0 or 1 fused benzo rings, wherein thephenyl or heterocycle is substituted by 0, 1, 2 or 3 substituentsselected from —R^(a), —OR^(a), —SR^(a), —NR^(a)R^(a), —OC(═O)R^(a),—NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a), —NR^(a)S(═O)₂R^(a), —S(═O)R^(a),—S(═O)₂R^(a), halogen, cyano, nitro and C₁₋₃alkyl substituted with 1-7halogen atoms.
 3. The compound according to claim 1, wherein R² isphenyl, —C(═O)phenyl, benzyl or a 5- or 6-membered ring heterocyclecontaining 1, 2 or 3 heteroatoms each independently selected from O, Nand S and additionally having 0 or 1 oxo groups and 0 or 1 fused benzorings, wherein the C₁₋₆alkyl, phenyl, benzyl or heterocycle issubstituted by 1, 2 or 3 substituents selected from —OR^(a), —SR^(a),—NR^(a)R^(a), —OC(═O)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen, cyano, nitro andC₁₋₃haloalkyl; and wherein the phenyl, —C(═O)phenyl, benzyl orheterocycle is additionally substituted by 0, 1 or 2 substituentsselected from C₁₋₆alkyl, phenyl and benzyl.
 4. The compound according toclaim 1, wherein R³ is C₁₋₆alkyl, —(CH₂)_(m)phenyl or—(CH₂)_(m)heterocycle, wherein the heterocycle is a 5- or 6-memberedring heterocycle containing 1, 2 or 3 heteroatoms each independentlyselected from O, N and S and additionally having 0 or 1 oxo groups and 0or 1 fused benzo rings.
 5. The compound according to claim 1, wherein R⁴is halogen, C₁₋₆alkyl, C₁₋₆haloalkyl, —(CH₂)_(m)phenyl or—(CH₂)_(m)heterocycle, wherein the heterocycle is a 5- or 6-memberedring heterocycle containing 1, 2 or 3 heteroatoms each independentlyselected from O, N and S and additionally having 0 or 1 oxo groups and 0or 1 fused benzo rings.
 6. The compound according to claim 1, wherein R⁵is C₁₋₆alkyl, —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O) NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl; or R⁵ is C₁₋₃alkyl containing 1 or 2substituents selected from —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),—OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano and nitro.
 7. The compound according to claim 1, wherein R⁶ is—R^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl.
 8. The compound according to claim 1,wherein R⁷ is —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a), —OC(═O)R^(a),—C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl.
 9. The compound according to claim 1,wherein R⁸ is C₁₋₆alkyl, —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),—OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano, nitro or C₁₋₃haloalkyl; or R⁸ is C₁₋₃alkyl containing 1 or 2substituents selected from —OR^(a), —SR^(a), —NR^(a)R^(a), —CO₂R^(a),—OC(═O)R^(a), —C(═O)NR^(a)R^(a), —NR^(a)C(═O)R^(a), —NR^(a)S(═O)R^(a),—NR^(a)S(═O)₂R^(a), —C(═O)R^(a), —S(═O)R^(a), —S(═O)₂R^(a), halogen,cyano and nitro.
 10. The compound according to claim 1, wherein R⁶ isOH.
 11. The compound according to claim 1 wherein: R³ is H; R⁴ is H; andR⁶ is OH.
 12. The compound according to any one of claims 1-11, whereinthe compound satisfies the equation:(K _(iαA) /K _(iβA))/(K _(iαE) /K _(iβE))>100, wherein K_(iαA) is theK_(i) value for the agonist in ER-α; K_(iβA) is the K_(i) value for theagonist in ER-β; K_(iαE) is the K_(i) value for estrogen in ER-α; andK_(iβE) is the K_(i) value for estrogen in ER-β.
 13. (canceled).
 14. Amethod for the treatment or prophylaxis of Alzheimer's disease, anxietydisorders, depressive disorders, osteoporosis, cardiovascular disease,rheumatoid arthritis or prostate cancer, which comprises administeringto a patient in need of such treatment or prophylaxis an effectiveamount of a compound according by any one of claims 1-11.
 15. Apharmaceutical composition comprising: a therapeutically-effectiveamount of a compound according to any one of claims 1-11; and apharmaceutically-acceptable diluent or carrier.
 16. A method for thetreatment or prophylaxis of Alzheimer's disease, anxiety disorders,depressive disorders, osteoporosis, cardiovascular disease, rheumatoidarthritis or prostate cancer, which comprises administering to a patientin need of such treatment or prophylaxis an effective amount of acompound according to claim
 12. 17. A pharmaceutical compositioncomprising: a therapeutically-effective amount of a compound accordingto claim 12; and a pharmaceutically-acceptable diluent or carrier.